RAIDD, a novel death adaptor molecule

ABSTRACT

The present invention relates to a novel molecule involved in the process of apoptosis. In particular, isolated nucleic acid molecules are provided encoding the human RAIDD protein and splice variants thereof—referred to as RAIDD-SV1 and RAIDD-SV2. RAIDD polypeptides are also provided as are vectors, host cells and recombinant methods for producing the same. The invention further relates to screening methods for identifying agonists and antagonists of RAIDD activity.

[0001] This application is a divisional of and claims priority under 35U.S.C. §120 to application Ser. No. 09/545,605, filed Apr. 7, 2000(allowed), which is a divisional of U.S. application Ser. No.08/995,159, filed Dec. 19, 1997 (now U.S. Pat. No. 6,130,079), whichclaims priority under 35 U.S.C. §119(e) of provisional application No.60/033,868, filed on Dec. 20, 1996, all of which is herein incorporatedby reference.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates to a novel molecule involved in theprocess of apoptosis. More specifically, isolated nucleic acid moleculesare provided encoding the human RAIDD protein and two splice variantsthereof—referred to as RAIDD-SV1 and RAIDD-SV2. RAIDD polypeptides arealso provided, as are vectors, host cells and recombinant methods forproducing the same. The invention further relates to screening methodsfor identifying agonists and antagonists of RAIDD activity.

[0004] 2. Related Art

[0005] Apoptosis, or programmed cell death, is a physiological processessential to the normal development and homeostasis of multicellularorganisms (Steller, Science 267:1445-1449 (1995)). Derangements ofapoptosis contribute to the pathogenesis of several human diseasesincluding cancer, neurodegenerative disorders, and acquired immunedeficiency syndrome (Thompson, Science 267:1456-1462 (1995)).

[0006] Apoptotic cell death is a multi-phase process (Vaux and Strasser,Proc. Natl. Acad. Sci. USA 93:2239-2244 (1996); Takahashi and Earnshaw,Curr. Opin. Genet. Dev. 6:50-55 (1996)). In the first phase cells reactto either external or internal stimuli capable of inducing apoptosisfollowed by transduction of the stimulus to the cell death effectormachinery. In the next phase, cell death machinery is activatedresulting in the cells undergoing dramatic changes in structure. Thislast phase is characterized by the activation of both nucleases andproteases.

[0007] Two cell surface receptors, CD95 (Fas/APO-1) and TNFR-1, havebeen shown to trigger apoptosis by their natural ligands or specificagonist antibodies (Baglioni, Tumor Necrosis Factors, “The Molecules andtheir Emerging Role in Medicine,” B. Beutler, ed., pp.425-438 (1992);Itoh et al., Cell 66:233-43 (1991); Trauth et al., Science 245:301-05(1989)). Both death receptors are members of the tumor necrosis factor(TNF)/nerve growth factor (NGF) receptor family which also includeTNFR-2, low-affinity MGFR, CD40 and CD30, among others (Smith et al.,Science 248:1019-23 (1990); Tewari et al., Cell 81:801-809 (1995)).

[0008] CD95 and TNFR-1 share a region of homology, appropriatelydesigned the “death domain,” required to signal apoptosis (Itoh et al.,J. Biol. Chem. 268:10932-7 (1993); Tartaglia et al., Cell 74:845-53(1993)). This shared “death domain” suggests that both receptorsinteract with a related set of signal transducing molecules that, untilrecently, remained unidentified. Using the two-hybrid system, threedeath domain-containing molecules, TRADD, FADD/MORT1 and RIP, wereisolated (Boldin et al., J. Biol. Chem. 270:7795-8 (1995); Chinnaiyan etal., Cell 81:505-12 (1995); Cleveland et al., Cell 81:479-82 (1995); Hsuet al., Cell 81:495-504 (1995); Stanger et al., Cell 81:513-23 (1995)).Subsequent studies showed that endogenous FADD associates with CD95 inan activation-dependent fashion (Kischkel et al., EMBO 14:5579-88(1995)), while similarly, endogenous TRADD and RIP were found complexedto activated TNFR-1 (Hsu et al., Cell 84:299-308 (1996); Hsu et al.,Immunity 4:387-96 (1996)). It has been postulated that TRADD acts as anadaptor molecule for TNFR-1 (Hsu et al., Cell 84:299-308 (1996)),mediating the interaction of TNFR-1 with FADD, while, by contrast, RIPmay be involved in NF-kB signaling (Hsu et al., Immunity 4:387-96(1996)). A dominant negative version of FADD (FADD-DN) blocks TNF- andCD95-induced apoptosis, suggesting that FADD functions as the commonsignaling conduit for cytokine-mediated cell death (Chinnaiyan et al.,J. Biol. Chem. 271:4961-65 (1996); Hsu et al., Cell 84:299-308 (1996)).

[0009] Overexpression of several proteins containing death domains hasbeen shown to result in the induction of apoptotic cell death in theabsence of an apoptotic induction stimulus (Vaux and Strasser, supra).Further, a number of viruses have been found to encode specificinhibitors of apoptosis, suggesting a role for apoptosis in antiviraldefense.

[0010] Overexpression of TRADD induces both apoptosis and NF-KBactivation-two of the most important activities signaled by TNFR-1 (Hsuet al., Cell 81:495-504 (1995)). Upon oligomerization of TNFR-1 bytrimeric TNF, TRADD is recruited to the receptor signaling complex (Hsuet al., Cell 84:299-308 (1996)). TRADD can then recruit the followingsignal transducing molecules: 1) TRAF2, a TNFR-2- and CD40- associatedmolecule (Rothe et al., Cell 78:681-92 (1994); Rothe et al., Science269:1424-1427 (1995)), that mediates NF-kB activation, 2) RIP,originally identified as a Fas/APO-1-interacting protein by two-hybridanalysis (Stanger et al., Cell 81:513-23 (1995)), that mediates NF-kBactivation and apoptosis (Hsu et al., Immunity 4:387-96 (1996)), and 3)FADD, a Fas/APO-1- associated molecule, that mediates apoptosis(Chinnaiyan et al., Cell 81:505-12 (1995); M. P. Boldin et al., J. Biol.Chem. 270:7795-8 (1995); F. C. Kischkel et al., EMBO 14:5579-5588(1995)).

[0011] Studies have demonstrated that FADD can recruit theICE/CED-3-like protease FLICE to the Fas/APO-1 death inducing signalingcomplex (Muzio et al., Cell 85:817-827 (1996); Boldin et al., Cell85:803-815 (1996)).

[0012] The first evidence for the involvement of ICE-like proteases inCD95- and TNFR-1 signaling came with the discovery that the poxvirusgene product CrmA blocks cell death triggered by both receptors (Enariet al., Nature 375:78-81 (1995); Los et al., Nature 375:81-3 (1995);Tewari et al., J. Biol. Chem. 270:3255-60 (1995)). In vitro, the serpinCrmA interacts only with the active forms of ICE and ICE-like proteases(Ray et al., Cell 69:597-604 (1992); Tewari et al., Cell 81:801-09(1995)). Yama and ICE-LAP3, two of the ICE-like enzymes most related toCED-3, are expressed as zymogens that are proteolytically activated uponligation of CD95 or TNFR-1 (Chinnaiyan et al., J. Biol. Chem.271:4961-65 (1996); Duan et al., J. Biol. Chem. 271:35013-35 (1996)).However, both Yama and ICE-LAP3 remained a proenzymes in anti-CD95treated CrmA-expressing cells, suggesting that CrmA inhibits an ICE-likeprotease upstream of Yama and ICE-LAP3 (Chinnaiyan et al., J. Biol.Chem. 271:4961-65 (1996)).

[0013] Phylogenetic analysis of the ICE/ced-3 gene family revealed threesubfamilies (Chinnaiyan et al., Current Biology 6:555-62 (1996); Duan etal., J. Biol. Chem. 271:35013-35 (1996)). Yama, ICE-LAP3, and Mch2 areclosely related to C. elegans Ced-3 and comprise the Ced-3 subfamily.ICE and the ICE-related genes, ICE rel II, and ICE rel III form the ICEsubfamily, while ICH-1 and its mouse homologue, NEDD-2 form the NEDD-2subfamily. Based on similarities with the structural prototypeinterleukin-1b converting enzyme, ICE/Ced-3 family members aresynthesized as zymogens that are capable of being processed to formactive heterodimeric enzymes (Thornberry et al., Nature 356:768-74(1992)).

SUMMARY OF THE INVENTION

[0014] The present invention provides isolated nucleic acid moleculescomprising a polynucleotide encoding the RAIDD polypeptide having theamino acid sequence shown in FIG. 1 (SEQ ID NO:2) or the amino acidsequence encoded by the cDNA clone deposited in a bacterial host as ATCCDeposit Number 97824 on Dec. 13, 1996.

[0015] The present invention further provides isolated nucleic acidmolecules comprising a polynucleotide encoding RAIDD polypeptide splicevariants—RAIDD-SV1 and RAIDD-SV2.

[0016] The present invention also relates to recombinant vectors, whichinclude the isolated nucleic acid molecules of the present invention,and to host cells containing the recombinant vectors, as well as tomethods of making such vectors and host cells and for using them forproduction of RAIDD polypeptides or peptides by recombinant techniques.

[0017] The invention further provides an isolated RAIDD polypeptide, andsplice variants thereof, having an amino acid sequence encoded by apolynucleotide described herein.

[0018] The present invention also provides a screening method foridentifying agonists and antagonists of RAIDD induced cell death, whichinvolves contacting cells which express the RAIDD protein with acandidate molecule and comparing the level of RAIDD induced cell deathto a standard level, the standard being assayed when contact is made inabsence of the candidate molecule; whereby, an increase in RAIDD inducedcell death in comparison to the standard indicates that the compound isan agonist and a decrease in RAIDD induced cell death in comparison tothe standard indicates that the compound is an antagonist.

[0019] The present invention further provides a screening method foridentifying compounds capable of interaction with the RAIDD polypeptidesof the present invention, which involves contacting a RAIDD polypeptidewith a candidate molecule and measuring whether the candidate moleculeinteracts with the RAIDD polypeptide.

BRIEF DESCRIPTION OF THE FIGURES

[0020]FIG. 1 shows the nucleotide (SEQ ID NO:1) and deduced amino acid(SEQ ID NO:2) sequences of RAIDD. The protein has a deduced molecularweight of about 22 kDa. The amino acid residues which are underlined anditalicized make up the RAIDD-SV1 splice variant. The amino acid residueswhich are italicized make up the RAIDD-SV2 splice variant.

[0021] FIGS. 2A-2C show a schematic representation of the RAIDD proteinand a comparison of regions of homology between the functional domainsof the RAIDD polypeptide and known proteins. FIG. 2A shows a schematicrepresentation of the domains of the RAIDD polypeptide. FIG. 2B shows acomparison between amino acid residues 9-78 of the RAIDD protein andportions of both ICH-1 (SEQ ID NO:3) and the C. elegans death proteaseCED-3 (SEQ ID NO:4). FIG. 2C shows a comparison between amino acidresidues 123-194 of the RAIDD protein and the death domain containingregions of FADD (SEQ ID NO:5), TRADD (SEQ ID NO:6), and RIP (SEQ IDNO:7). Identical amino acids in FIG. 2B and FIG. 2C are boxed. Numbersin FIGS. 2A-2C represent amino acid residues which correspond to aminoacids residues shown in SEQ ID NO:2.

[0022]FIG. 3 shows an analysis of the RAIDD amino acid sequence. Alpha,beta, turn and coil regions; hydrophilicity and hydrophobicity;amphipathic regions; flexible regions; antigenic index and surfaceprobability are shown. In the “Antigenic Index—Jameson-Wolf” graph,amino acid residues about 61 to about 71, about 112 to about 134, about144 to about 159, and about 168 to about 178, in FIG. 1 (SEQ ID NO:2)correspond to the shown highly antigenic regions of the RAIDD protein.

[0023]FIG. 4 shows the result of mutational analysis of the N-terminaldomain (NTD) in RAIDD and ICH-1. In vitro translated ³⁵S-labeled testproteins were incubated with either His-tagged RAIDD immobilized onto Nibeads or ICH-1 proteins bound to protein G-Sepharose. Alignment showsthe regions of sequence similarity that surround the two knowninactivating mutations in the prodomain of ced-3 (n1040 and n718,asterisked). Amino acids contained within each segment are numbered.Point mutations are shown as altered amino acid residues aligned to thecorresponding wild type amino acid. + indicates significant bindingwhile − indicates binding that was no different from background.

[0024]FIG. 5 shows that overexpression of RAIDD induces apoptosis whichis inhibitable by CrmA, the broad spectrum ICE family inhibitorz-VAD-fmk, and a catalytically inactive version of ICH-1 (Cys₃₀₂ toAla). The data (mean plus or minus standard error of the mean) shown arethe percentage of apoptotic cells as a function of the total number ofcells counted.

DETAILED DESCRIPTION

[0025] The present invention provides isolated nucleic acid moleculescomprising a polynucleotide encoding a RAIDD polypeptide having theamino acid sequence shown in SEQ ID NO:2, which was determined bysequencing a cloned cDNA. Regions of the RAIDD proteins of the presentinvention share sequence homology with human ICH-1 (SEQ ID NO:3), CED-3of C. elegans (SEQ ID NO:4), FADD (SEQ ID NO:5), TRADD (SEQ ID NO:6),and RIP (SEQ ID NO:7) (see FIGS. 2B and 2C). The nucleotide sequenceshown in SEQ ID NO:1 was obtained by sequencing the HSDME38XX clone,which was deposited on Dec. 13, 1996 at the American Type CultureCollection, 10801 University Blvd., Manassas, Va. 20110-2209, U.S.A.,and given accession number 97824. The deposited clone is inserted in thepBluescript SK(−) plasmid (Stratagene, LaJolla, Calif.) using the EcoRIand XhoI restriction endonuclease cleavage sites.

[0026] Splice variants of the RAIDD protein, referred to herein asRAIDD-SV1 and RAIDD-SV2, have been identified and included in thepresent invention. As used herein the phrase “splice variant” refers tocDNA molecules produced from RNA molecules initially transcribed fromthe same genomic DNA sequence which have undergone alternative RNAsplicing. Alternative RNA splicing occurs when a primary RNA transcriptundergoes splicing, generally for the removal of introns, which resultsin the production of more than one mRNA molecule each of which mayencode different amino acid sequences. The term “splice variant” alsorefers to the proteins encoded by the above cDNA molecules.

[0027] Nucleic Acid Molecules

[0028] Unless otherwise indicated, all nucleotide sequences determinedby sequencing a DNA molecule herein were determined using an automatedDNA sequencer (such as the Model 373 from Applied Biosystems, Inc.), andall amino acid sequences of polypeptides encoded by DNA moleculesdetermined herein were predicted by translation of a DNA sequencedetermined as above. Therefore, as is known in the art for any DNAsequence determined by this automated approach, any nucleotide sequencedetermined herein may contain some errors. Nucleotide sequencesdetermined by automation are typically at least about 90% identical,more typically at least about 95% to at least about 99.9% identical tothe actual nucleotide sequence of the sequenced DNA molecule. The actualsequence can be more precisely determined by other approaches includingmanual DNA sequencing methods well known in the art. As is also known inthe art, a single insertion or deletion in a determined nucleotidesequence compared to the actual sequence will cause a frame shift intranslation of the nucleotide sequence such that the predicted aminoacid sequence encoded by a determined nucleotide sequence will becompletely different from the amino acid sequence actually encoded bythe sequenced DNA molecule, beginning at the point of such an insertionor deletion.

[0029] Using the information provided herein, such as the nucleotidesequence in SEQ ID NO:1, a nucleic acid molecule of the presentinvention encoding a RAIDD polypeptide may be obtained using standardcloning and screening procedures, such as those for cloning cDNAs usingmRNA as starting material. Illustrative of the invention, the nucleicacid molecule described in SEQ ID NO:1 was discovered in human K562 cellline cDNA library. As noted in Example 4, the RAIDD gene isconstitutively expressed in all adult and fetal tissues examined withsubstantial expression in each. These tissues include heart, testis,skeletal muscle, liver and kidney. The determined nucleotide sequence ofthe RAIDD cDNA of SEQ ID NO:1 contains an open reading frame encoding aprotein of about 199 amino acid residues and a deduced molecular weightof about 22 kDa. The amino acid sequence of the predicted full lengthRAIDD polypeptide is shown in SEQ ID NO:2. The N-terminal portion of theRAIDD protein shown in SEQ ID NO:2 (amino acid residues from about 8 toabout 80) is about 40% identical and about 50% similar to the pro-domainof the human cysteine protease ICH-1 (amino acid residues 23 to 91 inFIG. 2B) (SEQ ID NO:3) and about 35% identical and about 40% similar tothe pro-domain of the C. elegans death protease CED-3 (amino acidresidues 10 to 77 in FIG. 2B) (SEQ ID NO:4). In addition, the C-terminalportion of the RAIDD protein shown in SEQ ID NO:2 (amino acids residuesfrom about 123 to about 194) is about 25% identical and about 35%similar to the death domain region of the human FADD protein (amino acidresidues 104 to 177 in FIG. 2C) (SEQ ID NO:5), about 25% identical andabout 35% similar to the death domain region of the human TRADD protein(amino acid residues 222 to 302 in FIG. 2C) (SEQ ID NO:6), and about 30%identical and about 30% similar to the death domain region of the humanRIP protein (amino acid residues 590 to 666 in FIG. 2C) (SEQ ID NO:7).

[0030] As one of ordinary skill would appreciate, due to thepossibilities of sequencing errors, the predicted RAIDD polypeptideencoded by the deposited cDNA comprises about 199 amino acids, but maybe anywhere in the range of about 189 to about 209 amino acids.

[0031] Splice variants of RAIDD have also been identified. These splicevariants include RAIDD-SV1 and RAIDD-SV2. RAIDD-SV1 comprisesessentially the linker region and death domain of the full-length RAIDDpolypeptide (amino acid residues from about 99 to about 199 shown in SEQID NO:2). RAIDD-SV2 comprises essentially only the death domain of thefull-length RAIDD polypeptide (amino acid residues from about 118 toabout 199 shown in SEQ ID NO:2).

[0032] As indicated, nucleic acid molecules of the present invention maybe in the form of RNA, such as mRNA, or in the form of DNA, including,for instance, cDNA and genomic DNA obtained by cloning or producedsynthetically. The DNA may be double-stranded or single-stranded.Single-stranded DNA or RNA may be the coding strand, also known as thesense strand, or it may be the non-coding strand, also referred to asthe anti-sense strand.

[0033] By “isolated” nucleic acid molecule(s) is intended a nucleic acidmolecule, DNA or RNA, which has been removed from its native environmentFor example, recombinant DNA molecules contained in a vector areconsidered isolated for the purposes of the present invention. Furtherexamples of isolated DNA molecules include recombinant DNA moleculesmaintained in heterologous host cells or purified (partially orsubstantially) DNA molecules in solution. Isolated RNA molecules includein vivo or in vitro RNA transcripts of the DNA molecules of the presentinvention. Isolated nucleic acid molecules according to the presentinvention further include such molecules produced synthetically.

[0034] Isolated nucleic acid molecules of the present invention includeDNA molecules comprising the open reading frame (ORF) shown in SEQ IDNO:1; DNA molecules comprising the ORF shown in SEQ ID NO:1 without theATG codon encoding the N terminal methionine residue; DNA moleculesencoding amino acid residues from about 99 to about 199 in SEQ ID NO:2;DNA molecules encoding amino acid residues from about 118 to about 199in SEQ ID NO:2; and DNA molecules which comprise a sequencesubstantially different from those described above but which, due to thedegeneracy of the genetic code, still encode the RAIDD protein or asplice variant thereof. Of course, the genetic code is well known in theart. Thus, it would be routine for one skilled in the art to generatesuch degenerate variants.

[0035] In another aspect, the invention provides isolated nucleic acidmolecules encoding the RAIDD polypeptide having an amino acid sequenceencoded by the cDNA clone contained in the plasmid deposited as ATCCDeposit No. 97824 on Dec. 13, 1996. The invention further provides anisolated nucleic acid molecule having the nucleotide sequence shown inSEQ ID NO:1 or the nucleotide sequence of the RAIDD cDNA contained inthe above-described deposited clone, or a nucleic acid molecule having asequence complementary to one of the above sequences. Such isolatedmolecules, particularly DNA molecules, are useful as probes for genemapping, by in situ hybridization with chromosomes, and for detectingexpression of the RAIDD gene in human tissue, for instance, by Northernblot analysis.

[0036] The present invention is further directed to fragments of theisolated nucleic acid molecules described herein. By a fragment of anisolated nucleic acid molecule having the nucleotide sequence of thedeposited cDNA or the nucleotide sequence shown in SEQ ID NO:1 isintended fragments at least about 15 nt, and more preferably at leastabout 20 nt, still more preferably at least about 30 nt, and even morepreferably, at least about 40 nt in length which are useful asdiagnostic probes and primers as discussed herein. Of course larger DNAfragments 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600,650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, or 1125 nt inlength of the sequence shown in SEQ ID NO:1 are also useful according tothe present invention as are fragments corresponding to most, if notall, of the nucleotide sequence of the cDNA clone contained in theplasmid deposited as ATCC Deposit No. 97824 or as shown in SEQ ID NO:1.By a fragment at least 20 nt in length, for example, is intendedfragments which include 20 or more contiguous bases from the nucleotidesequence of the deposited cDNA or the nucleotide sequence as shown inSEQ ID NO:1.

[0037] Preferred nucleic acid fragments of the present invention includenucleic acid molecules encoding epitope-bearing portions of the RAIDDprotein. In particular, such nucleic acid fragments of the presentinvention include nucleic acid molecules encoding: a polypeptidecomprising amino acid residues from about 61 to about 71 in SEQ ID NO:2;a polypeptide comprising amino acid residues from about 112 to about 134in SEQ ID NO:2; a polypeptide comprising amino acid residues from about144 to about 159 in SEQ ID NO:2; and a polypeptide comprising amino acidresidues from about 168 to about 178 in SEQ ID NO:2. The inventors havedetermined that the above polypeptide fragments are antigenic regions ofthe RAIDD protein. Methods for determining other such epitope-bearingportions of the RAIDD protein are described in detail below.

[0038] In another aspect, the invention provides an isolated nucleicacid molecule comprising a polynucleotide which hybridizes understringent hybridization conditions to a portion of the polynucleotide ina nucleic acid molecule of the invention described above, for instance,the cDNA clone contained in ATCC Deposit 97824. By “stringenthybridization conditions” is intended overnight incubation at 42° C. ina solution comprising: 50% formamide, 5×SSC (750 mM NaCl, 75 mMtrisodium citrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt'ssolution, 10% dextran sulfate, and 20 μg/ml 1 denatured, sheared salmonsperm DNA, followed by washing the filters in 0.1×SSC at about 65° C.

[0039] By a polynucleotide which hybridizes to a “portion” of apolynucleotide is intended a polynucleotide (either DNA or RNA)hybridizing to at least about 15 nucleotides (nt), and more preferablyat least about 20 nt, still more preferably at least about 30 nt, andeven more preferably about 30-70 nt of the reference polynucleotide.These are useful as diagnostic probes and primers as discussed above andin more detail below.

[0040] By a portion of a polynucleotide of “at least 20 nt in length,”for example, is intended 20 or more contiguous nucleotides from thenucleotide sequence of the reference polynucleotide (e.g., the depositedcDNA or the nucleotide sequence as shown in SEQ ID NO:1). Of course, apolynucleotide which hybridizes only to a poly A sequence (such as the3′ terminal poly(A) tract of the RAIDD cDNA shown in SEQ ID NO:1), or toa complementary stretch of T (or U) resides, would not be included in apolynucleotide of the invention used to hybridize to a portion of anucleic acid of the invention, since such a polynucleotide wouldhybridize to any nucleic acid molecule containing a poly (A) stretch orthe complement thereof (e.g., practically any double-stranded cDNAclone).

[0041] As indicated, nucleic acid molecules of the present inventionwhich encode a RAIDD polypeptide may include, but are not limited tothose encoding the amino acid sequence of the mature polypeptide, byitself; the coding sequence for the mature polypeptide and additionalsequences, such as those encoding an amino acid leader or secretorysequence, such as a pre-, or pro- or prepro-protein sequence; the codingsequence of the mature polypeptide, with or without the aforementionedadditional coding sequences, together with additional, non-codingsequences, including for example, but not limited to introns andnon-coding 5′ and 3′ sequences, such as the transcribed, non-translatedsequences that play a role in transcription, mRNA processing, includingsplicing and polyadenylation signals, for example—ribosome binding andstability of mRNA; an additional coding sequence which codes foradditional amino acids, such as those which provide additionalfunctionalities. Thus, the sequence encoding the polypeptide may befused to a marker sequence, such as a sequence encoding a peptide whichfacilitates purification of the fused polypeptide. In certain preferredembodiments of this aspect of the invention, the marker amino acidsequence is a hexa-histidine peptide, such as the tag provided in a pQEvector (Qiagen, Inc.), among others, many of which are commerciallyavailable. As described in Gentz et al., Proc. Natl. Acad. Sci. USA86:821-824 (1989), for instance, hexa-histidine provides for convenientpurification of the fusion protein. The “HA” tag is another peptideuseful for purification which corresponds to an epitope derived from theinfluenza hemagglutinin protein, which has been described by Wilson etal., Cell 37:767 (1984). As discussed below, other such fusion proteinsinclude the RAIDD protein fused to Fc at the N- or C-terminus.

[0042] The present invention further relates to variants of the nucleicacid molecules of the present invention, which encode portions, analogsor derivatives of a RAIDD protein. Variants may occur naturally, such asa natural allelic variant. By an “allelic variant” is intended one ofseveral alternate forms of a gene occupying a given locus on achromosome of an organism. Genes II, Lewin, B., ed., John Wiley & Sons,New York (1985). Non-naturally occurring variants may be produced usingart-known mutagenesis techniques.

[0043] Such variants include those produced by nucleotide substitutions,deletions or additions, which may involve one or more nucleotides. Thevariants may be altered in coding regions, non-coding regions, or both.Alterations in the coding regions may produce conservative ornon-conservative amino acid substitutions, deletions or additions.Especially preferred among these are silent substitutions, additions anddeletions, which do not alter the properties and activities of the RAIDDprotein or portions thereof. Also especially preferred in this regardare conservative substitutions.

[0044] Further embodiments of the invention include isolated nucleicacid molecules comprising a polynucleotide having a nucleotide sequenceat least 90% identical, and more preferably at least 95%, 96%, 97%, 98%or 99% identical to (a) a nucleotide sequence encoding the RAIDDpolypeptide having the amino acid sequence in SEQ ID NO:2; (b) anucleotide sequence encoding the RAIDD polypeptide having the aminosequence shown in SEQ ID NO:2 but lacking the N-terminal methionine(amino acid residues 2 to 199 in SEQ ID NO:2); (c) a nucleotide sequenceencoding the RAIDD-SV1 protein (amino acid residues from about 99 toabout 199 in SEQ ID NO:2); (d) a nucleotide sequence encoding theRAIDD-SV2 protein (amino acid residues from about 118 to about 199 inSEQ ID NO:2); (e) a nucleotide sequence encoding a RAIDD cysteineprotease domain (amino acid residues from about 8 to about 80 in SEQ IDNO:2); (f) a nucleotide sequence encoding a RAIDD death domain (aminoacid residues from about 123 to about 194 in SEQ ID NO:2); (g) anucleotide sequence encoding the RAIDD polypeptide having the amino acidsequence encoded by the cDNA clone contained in ATCC Deposit No. 97824;or (h) a nucleotide sequence complementary to any of the nucleotidesequences in (a), (b), (c), (d), (e), (f) or (g).

[0045] By a polynucleotide having a nucleotide sequence at least, forexample, 95% “identical” to a reference nucleotide sequence encoding aRAIDD polypeptide is intended that the nucleotide sequence of thepolynucleotide is identical to the reference sequence except that thepolynucleotide sequence may include up to five point mutations per each100 nucleotides of the reference nucleotide sequence encoding the RAIDDpolypeptide. In other words, to obtain a polynucleotide having anucleotide sequence at least 95% identical to a reference nucleotidesequence, up to 5% of the nucleotides in the reference sequence may bedeleted or substituted with another nucleotide, or a number ofnucleotides up to 5% of the total nucleotides in the reference sequencemay be inserted into the reference sequence. These mutations of thereference sequence may occur at the 5′ or 3′ terminal positions of thereference nucleotide sequence or anywhere between those terminalpositions, interspersed either individually among nucleotides in thereference sequence or in one or more contiguous groups within thereference sequence.

[0046] As a practical matter, whether any particular nucleic acidmolecule is at least 90%,95%,96%, 97%, 98% or 99% identical to thosedescribed above can be determined conventionally using known computerprograms such as the Bestfit program (Wisconsin Sequence AnalysisPackage, Version 8 for Unix, Genetics Computer Group, UniversityResearch Park, 575 Science Drive, Madison, Wis. 53711). Bestfit uses thelocal homology algorithm of Smith and Waterman, Advances in AppliedMathematics 2:482-489 (1981), to find the best segment of homologybetween two sequences. When using Bestfit or any other sequencealignment program to determine whether a particular sequence is, forinstance, 95% identical to a reference sequence according to the presentinvention, the parameters are set, of course, such that the percentageof identity is calculated over the full length of the referencenucleotide sequence and that gaps in homology of up to 5% of the totalnumber of nucleotides in the reference sequence are allowed.

[0047] The present application is directed to nucleic acid molecules atleast 90%, 95%, 96%, 97%, 98% or 99% identical to those described aboveirrespective of whether they encode a polypeptide having RAIDD activity.This is because even where a particular nucleic acid molecule does notencode a polypeptide having RAIDD activity, one of skill in the artwould still know how to use the nucleic acid molecule, for instance, asa hybridization probe or a polymerase chain reaction (PCR) primer. Usesof the nucleic acid molecules of the present invention that do notencode a polypeptide having RAIDD activity include, inter alia, (1)isolating the RAIDD gene, or splice or allelic variants thereof, in acDNA library; (2) in situ hybridization (e.g., “FISH”) to metaphasechromosomal spreads to provide precise chromosomal location of the RAIDDgene, as described in Verma et al., Human Chromosomes: A Manual of BasicTechniques, Pergamon Press, New York (1988); and (3) Northern Blotanalysis for detecting RAIDD mRNA expression in specific tissues.

[0048] Preferred, however, are nucleic acid molecules having sequencesat least 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acidsequence shown in SEQ ID NO:1 or to the nucleic acid sequence of thedeposited cDNA which do, in fact, encode a polypeptide having RAIDDprotein activity. By “a polypeptide having RAIDD activity” is intendedpolypeptides exhibiting activity similar, but not necessarily identical,to an activity of the RAIDD protein of the invention (either thefull-length protein or splice variants thereof), as measured in aparticular biological assay. For example, RAIDD protein activity can bemeasured using either the ICH-1 binding or apoptosis assay described inExample 4.

[0049] Of course, due to the degeneracy of the genetic code, one ofordinary skill in the art will immediately recognize that a large numberof the nucleic acid molecules having a sequence at least 90%, 95%, 96%,97%, 98%, or 99% identical to the nucleic acid sequence of the depositedcDNA or the nucleic acid sequence shown in SEQ ID NO:1 will encode apolypeptide “having RAIDD protein activity.” In fact, since degeneratevariants of these nucleotide sequences all encode the same polypeptide,this will be clear to the skilled artisan even without performing theabove described comparison assays. It will be further recognized in theart that, for such nucleic acid molecules that are not degeneratevariants, a reasonable number will also encode a polypeptide havingRAIDD protein activity. This is because the skilled artisan is fullyaware of amino acid substitutions that are either less likely or notlikely to significantly effect protein function (e.g., replacing onealiphatic amino acid with a second aliphatic amino acid).

[0050] For example, guidance concerning how to make phenotypicallysilent amino acid substitutions is provided in Bowie, J. U. et al.,“Deciphering the Message in Protein Sequences: Tolerance to Amino AcidSubstitutions,” Science 247:1306-1310 (1990), wherein the authorsindicate that proteins are surprisingly tolerant of amino acidsubstitutions.

[0051] Vectors and Host Cells

[0052] The present invention also relates to vectors which include theisolated DNA molecules of the present invention, host cells which aregenetically engineered with the recombinant vectors, and the productionof RAIDD polypeptides or fragments thereof by recombinant techniques.

[0053] The polynucleotides may be joined to a vector containing aselectable marker for propagation in a host. Generally, a plasmid vectoris introduced in a precipitate, such as a calcium phosphate precipitate,or in a complex with a charged lipid. If the vector is a virus, it maybe packaged in vitro using an appropriate packaging cell line and thentransduced into host cells.

[0054] The DNA insert should be operatively linked to an appropriatepromoter, such as the phage lambda PL promoter, the E. coli lac, trp andtac promoters, the SV40 early and late promoters and promoters ofretroviral LTRs, to name a few. Other suitable promoters will be knownto the skilled artisan. The expression constructs will further containsites for transcription initiation, termination and, in the transcribedregion, a ribosome binding site for translation. The coding portion ofthe transcripts expressed by the constructs will preferably include atranslation initiating at the beginning and a termination codon (UAA,UGA or UAG) appropriately positioned at the end of the polypeptide to betranslated.

[0055] As indicated, the expression vectors will preferably include atleast one selectable marker. Such markers include dihydrofolatereductase or neomycin resistance for eukaryotic cell culture andtetracycline or ampicillin resistance genes for culturing in E. coli andother bacteria. Representative examples of appropriate hosts include,but are not limited to, bacterial cells, such as E. coli, Streptomycesand Salmonella typhimurium cells; fungal cells, such as yeast cells;insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animalcells such as CHO, COS and Bowes melanoma cells; and plant cells.Appropriate culture mediums and conditions for the above-described hostcells are known in the art.

[0056] Among vectors preferred for use in bacteria include pQE70, pQE60and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors,Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available fromStratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 availablefrom Pharmacia. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT,pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG andpSVL available from Pharmacia. Other suitable vectors will be readilyapparent to the skilled artisan.

[0057] Introduction of the construct into the host cell can be effectedby calcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection or other methods. Such methods are described in many standardlaboratory manuals, such as Davis et al., Basic Methods In MolecularBiology (1986).

[0058] The polypeptide may be expressed in a modified form, such as afusion protein, and may include not only secretion signals, but alsoadditional heterologous functional regions. For instance, a region ofadditional amino acids, particularly charged amino acids, may be addedto the N-terminus of the polypeptide to improve stability andpersistence in the host cell, during purification, or during subsequenthandling and storage. Also, peptide moieties may be added to thepolypeptide to facilitate purification. Such regions may be removedprior to final preparation of the polypeptide. The addition of peptidemoieties to polypeptides to engender secretion or excretion, to improvestability and to facilitate purification, among others, are familiar androutine techniques in the art. A preferred fusion protein comprises aheterologous region from immunoglobulin that is useful to solubilizeproteins. For example, EP-A-O 464 533 (Canadian counterpart 2045869)discloses fusion proteins comprising various portions of constant regionof immunoglobin molecules together with another human protein or partthereof. In many cases, the Fe part in a fusion protein is thoroughlyadvantageous for use in therapy and diagnosis and thus results, forexample, in improved pharmacokinetic properties (EP-A 0232 262). On theother hand, for some uses it would be desirable to be able to delete theFc part after the fusion protein has been expressed, detected andpurified in the advantageous manner described. This is the case when Fcportion proves to be a hindrance to use in therapy and diagnosis, forexample when the fusion protein is to be used as antigen forimmunizations. In drug discovery, for example, human proteins have beenfused with Fc portions for the purpose of high-throughput screeningassays to identify molecules with antagonistic activity. One example isthe hIL-5 receptor which has been used to identify antagonists of hIL-5.See, D. Bennett et al., Jour. of Molecular Recognition, 8:52-58 (1995)and K. Johanson et al., Jour. Biol. Chem., 270:9459-9471 (1995).

[0059] The RAIDD protein can be recovered and purified from recombinantcell cultures by well-known methods including ammonium sulfate orethanol precipitation, acid extraction, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography, hydroxylapatite chromatographyand lectin chromatography. Most preferably, high performance liquidchromatography (“HPLC”) is employed for purification. Polypeptides ofthe present invention include naturally purified products, products ofchemical synthetic procedures, and products produced by recombinanttechniques from a prokaryotic or eukaryotic host, including, forexample, bacterial, yeast, higher plant, insect and mammalian cells.Depending upon the host employed in a recombinant production procedure,the polypeptides of the present invention may be glycosylated or may benon-glycosylated. In addition, polypeptides of the invention may alsoinclude an initial modified methionine residue, in some cases as aresult of host-mediated processes.

[0060] RAIDD Polypeptides and Fragments

[0061] The invention further provides an isolated RAIDD polypeptidehaving the amino acid sequence encoded by the deposited cDNA, or theamino acid sequence in SEQ ID NO:2, or a peptide or polypeptidecomprising a portion of the above polypeptides.

[0062] It will be recognized in the art that some amino acid sequencesof the RAIDD polypeptide can be varied without significant effect of thestructure or function of the protein. If such differences in sequenceare contemplated, it should be remembered that there will be criticalareas on the protein which determine activity.

[0063] Thus, the invention further includes variations of the RAIDDpolypeptides of the present invention which show substantial RAIDDpolypeptide activity or which include regions of a RAIDD protein such asthe protein portions discussed below. Such mutants include deletions,insertions, inversions, repeats, and type substitutions. As indicatedabove, guidance concerning which amino acid changes are likely to bephenotypically silent can be found in Bowie, J. U. et al., “Decipheringthe Message in Protein Sequences: Tolerance to Amino AcidSubstitutions,” Science 247:1306-1310 (1990).

[0064] Thus, the fragment, derivative or analog of the polypeptide ofSEQ ID NO:2, or that encoded by the deposited cDNA, may be (i) one inwhich one or more of the amino acid residues are substituted with aconserved or non-conserved amino acid residue (preferably a conservedamino acid residue) and such substituted amino acid residue may or maynot be one encoded by the genetic code, or (ii) one in which one or moreof the amino acid residues includes a substituent group, or (iii) one inwhich the polypeptide is fused with another compound, such as a compoundto increase the half-life of the polypeptide (for example, polyethyleneglycol), or (iv) one in which the additional amino acids are fused tothe mature polypeptide, such as an IgG Fc fusion region peptide orleader or secretory sequence or a sequence which is employed forpurification of the mature polypeptide or a proprotein sequence. Suchfragments, derivatives and analogs are deemed to be within the scope ofthose skilled in the art from the teachings herein.

[0065] Of particular interest are substitutions of charged amino acidswith another charged amino acid and with neutral or negatively chargedamino acids. The latter results in proteins with reduced positive chargeto improve the characteristics of the RAIDD protein. The prevention ofaggregation is highly desirable. Aggregation of proteins not onlyresults in a loss of activity but can also be problematic when preparingpharmaceutical formulations, because they can be immunogenic. (Pinckardet al., Clin Exp. Immunol. 2:331-340 (1967); Robbins et al., Diabetes36:838-845 (1987); Cleland et al., Crit. Rev. Therapeutic Drug CarrierSystems 10:307-377 (1993)).

[0066] As indicated, changes are preferably of a minor nature, such asconservative amino acid substitutions that do not significantly affectthe folding or activity of the protein (see Table 1). TABLE 1Conservative Amino Acid Substitutions. Aromatic Phenylalanine TryptophanTyrosine Hydrophobic Leucine Isoleucine Valine Polar GlutamineAsparagine Basic Arginine Lysine Histidine Acidic Aspartic Acid GlutamicAcid Small Alanine Serine Threonine Methionine Glycine

[0067] Of course, the number of amino acid substitutions a skilledartisan would make depends on many factors, including those describedabove and below. Generally speaking, the number of substitutions for anygiven RAIDD polypeptide, or mutant thereof, will not be more than 50,40, 30, 20, 10, 5, or 3, depending on the objective.

[0068] Amino acids in a RAIDD protein of the present invention that areessential for function can be identified by methods known in the art,such as site-directed mutagenesis or alanine-scanning mutagenesis(Cunningham and Wells, Science 244:1081-1085 (1989)). The latterprocedure introduces single alanine mutations at every residue in themolecule. The resulting mutant molecules are then tested for biologicalactivity such as the ability to bind ICH-1 or CED-3 or the ability toinduce apoptosis when overexpressed. Sites that are critical forRAIDD-ligand interaction can also be determined by structural analysissuch as crystallization, nuclear magnetic resonance or photoaffinitylabeling (Smith et al., J. Mol. Biol. 224:899-904 (1992) and de Vos etal., Science 255:306-312 (1992)).

[0069] The polypeptides of the present invention are preferably providedin an isolated form. By “isolated polypeptide” is intended a polypeptideremoved from its native environment. Thus, a polypeptide produced and/orcontained within a recombinant host cell is considered isolated forpurposes of the present invention. Also intended as an “isolatedpolypeptide” are polypeptides that have been purified, partially orsubstantially, from a recombinant host cell. For example, arecombinantly produced version of a RAIDD polypeptide described hereincan be substantially purified by the one-step method described in Smithand Johnson, Gene 67:31-40 (1988).

[0070] The polypeptides of the present invention include the polypeptideencoded by the deposited cDNA, a polypeptide comprising amino acids fromabout 1 to about 199 in SEQ ID NO:2; a polypeptide comprising aminoacids from about 2 to about 199 in SEQ ID NO:2; a polypeptide comprisingamino acids from about 8 to about 80 in SEQ ID NO:2; a polypeptidecomprising amino acids from about 99 to about 199 in SEQ ID NO:2; apolypeptide comprising amino acids from about 118 to about 199 in SEQ IDNO:2; a polypeptide comprising amino acids from about 123 to about 194in SEQ ID NO:2; as well as polypeptides which are at least 90%identical, more preferably at least 95% identical, still more preferablyat least 96%, 97%, 98% or 99% identical to those described above andalso include portions of such polypeptides with at least 30 amino acidsand more preferably at least 50 amino acids.

[0071] By a polypeptide having an amino acid sequence at least, forexample, 95% “identical” to a reference amino acid sequence of a RAIDDpolypeptide is intended that the amino acid sequence of the polypeptideis identical to the reference sequence except that the polypeptidesequence may include up to five amino acid alterations per each 100amino acids of the reference amino acid of the RAIDD polypeptide. Inother words, to obtain a polypeptide having an amino acid sequence atleast 95% identical to a reference amino acid sequence, up to 5% of theamino acid residues in the reference sequence may be deleted orsubstituted with another amino acid, or a number of amino acids up to 5%of the total amino acid residues in the reference sequence may beinserted into the reference sequence. These alterations of the referencesequence may occur at the amino or carboxy terminal positions of thereference amino acid sequence or anywhere between those terminalpositions, interspersed either individually among residues in thereference sequence or in one or more contiguous groups within thereference sequence.

[0072] As a practical matter, whether any particular polypeptide is atleast 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, theamino acid sequence shown in SEQ ID NO:2 or to the amino acid sequenceencoded by deposited cDNA clone can be determined conventionally usingknown computer programs such the Bestfit program (Wisconsin SequenceAnalysis Package, Version 8 for Unix, Genetics Computer Group,University Research Park, 575 Science Drive, Madison, Wis. 53711). Whenusing Bestfit or any other sequence alignment program to determinewhether a particular sequence is, for instance, 95% identical to areference sequence according to the present invention, the parametersare set, of course, such that the percentage of identity is calculatedover the full length of the reference amino acid sequence and that gapsin homology of up to 5% of the total number of amino acid residues inthe reference sequence are allowed.

[0073] The polypeptides of the present invention could be used asmolecular weight markers on SDS-PAGE gels or on molecular sieve gelfiltration columns using methods well known to those of skill in theart.

[0074] In another aspect, the invention provides a peptide orpolypeptide comprising an epitope-bearing portion of a polypeptide ofthe invention. The epitope of this polypeptide portion is an immunogenicor antigenic epitope of a polypeptide described herein. An “immunogenicepitope” is defined as a part of a protein that elicits an antibodyresponse when the whole protein is the immunogen. On the other hand, aregion of a protein molecule to which an antibody can bind is defined asan “antigenic epitope.” The number of immunogenic epitopes of a proteingenerally is less than the number of antigenic epitopes. See, forinstance, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998-4002 (1983).

[0075] As to the selection of peptides or polypeptides bearing anantigenic epitope (i.e., that contain a region of a protein molecule towhich an antibody can bind), it is well known in that art thatrelatively short synthetic peptides that mimic part of a proteinsequence are routinely capable of eliciting an antiserum that reactswith the partially mimicked protein. See, for instance, Sutcliffe etal., Antibodies that react with predetermined sites on proteins. Science219:660-666 (1983). Peptides capable of eliciting protein-reactive seraare frequently represented in the primary sequence of a protein, can becharacterized by a set of simple chemical rules, and are confinedneither to immunodominant regions of intact proteins (i.e., immunogenicepitopes) nor to the amino or carboxyl terminals.

[0076] Antigenic epitope-bearing peptides and polypeptides of theinvention are therefore useful to raise antibodies, including monoclonalantibodies, that bind specifically to a polypeptide of the invention.See, for instance, Wilson et al., Cell 37:767-778 (1984) at 777.

[0077] Antigenic epitope-bearing peptides and polypeptides of theinvention preferably contain a sequence of at least seven, morepreferably at least nine and most preferably between about at leastabout 15 to about 30 amino acids contained within the amino acidsequence of a polypeptide of the invention.

[0078] Non-limiting examples of antigenic polypeptides or peptides thatcan be used to generate RAIDD-specific antibodies include: a polypeptidecomprising amino acid residues from about 61 to about 71 in SEQ ID NO:2;a polypeptide comprising amino acid residues from about 112 to about 134in SEQ ID NO:2; a polypeptide comprising amino acid residues from about144 to about 159 in SEQ ID NO:2; and a polypeptide comprising amino acidresidues from about 168 to about 178 in SEQ ID NO:2. As indicated above,the inventors have determined that the above polypeptide fragments areantigenic regions of the RAIDD protein shown in SEQ ID NO:2.

[0079] The epitope-bearing peptides and polypeptides of the inventionmay be produced by any conventional means. Houghten, R. A. (1985)General method for the rapid solid-phase synthesis of large numbers ofpeptides: specificity of antigen-antibody interaction at the level ofindividual amino acids. Proc. Natl. Acad. Sci. USA 82:5131-5135. This“Simultaneous Multiple Peptide Synthesis (SMPS)” process is furtherdescribed in U.S. Pat. No. 4,631,211 to Houghten et al., (1986).

[0080] As one of skill in the art will appreciate, the RAIDDpolypeptides of the present invention and the epitope-bearing fragmentsthereof described above can be combined with parts of the constantdomain of immunoglobulins (IgG), resulting in chimeric polypeptides.These fusion proteins facilitate purification and show an increasedhalf-life in vivo. This has been shown, e.g., for chimeric proteinsconsisting of the first two domains of the human CD4-polypeptide andvarious domains of the constant regions of the heavy or light chains ofmammalian immunoglobulins (EPA 394,827; Traunecker et al., Nature331:84-86 (1988)). Fusion proteins that have a disulfide-linked dimericstructure due to the IgG part can also be more efficient in binding andneutralizing other molecules than monomeric RAIDD proteins or proteinfragments alone (Fountoulakis et al., J. Biochem 270:3958-3964 (1995)).

[0081] Agonists and Antagonists of RAIDD Polypeptide Function

[0082] In one aspect, the present invention is directed to a method foridentifying molecules capable of inhibiting or enhancing RAIDD inducedcell death. The method involves contacting cells which express a RAIDDpolypeptide of the present invention with a candidate molecule,measuring the level of RAIDD induced cell death, and comparing the levelof cell death to a standard level of RAIDD induced cell death, whereinthe standard level of RAIDD induced cell death is assayed using cellsexpressing a RAIDD polypeptide in absence of the candidate molecule.According to this method, an increased level of RAIDD induced cell deathin comparison to the standard indicates that the candidate molecule isan agonist and a decreased level of RAIDD induced cell death incomparison to the standard indicates that the candidate molecule is anantagonist.

[0083] By “RAIDD induced cell death” is intended cell death induced by apathway involving the RAIDD polypeptide. Methods for measuring RAIDDinduced cell death include the RAIDD induced cell death/apoptotic assaysdescribed in Example 4. By a “candidate molecule” is intended anynaturally occurring or synthetic compound which is screened by themethod of the present invention for agonistic or antagonistic activitywith respect to an activity of a RAIDD polypeptide of the presentinvention (e.g., induction of RAIDD induced cell death and the abilityto bind cellular ligands such as RIP and ICH-1). A “candidate molecule”is also intended to refer to any molecule which is screened for bindingto a RAIDD polypeptide of the present invention. By “binding to a RAIDDpolypeptide” is intended an association between a RAIDD polypeptide andthe candidate molecule which results from an attractive molecularinteraction. By “agonist” is intended naturally occurring and syntheticcompounds capable of enhancing or potentiating an activity of a RAIDDpolypeptide of the present invention. By “antagonist” is intendednaturally occurring and synthetic compounds capable of inhibiting anactivity of a RAIDD polypeptide. Whether a candidate “agonist” or“antagonist” of the present invention can enhance or inhibit a RAIDDactivity can be determined using art-known assays and those assaysdescribed herein.

[0084] In a preferred embodiment, cells which express a RAIDDpolypeptide via an expression construct are used for the identificationof antagonists and agonists of RAIDD activity. In this embodiment, thecandidate molecule is either added exogenously or is producedendogenously by the transfected cells. Measurement of an increased ordecreased level of RAIDD induced cell death is made by comparing thepercentage of cells in contact with the candidate molecule which undergocell death to those of a standard in which the cells are not in contactwith the candidate molecule. As described in Example 4, expression ofP-galactosidase via a reporter construct may be used to identify cellswhich have undergone apoptotic demise.

[0085] In an additional embodiments, cells are transfected with one ormore expression constructs which express both RAIDD and the candidatemolecule. As above, the percentage of cells undergoing apoptotic cellsdeath is scored as in Example 4.

[0086] As noted in Example 4, several inhibitors of RAIDD induced celldeath have been identified using the method disclosed herein. Theseinclude the ICE peptide inhibitor z-VAD-fmk, CrmA and catalyticallyinactive ICH-1. Other potential agonists and antagonists of the presentinvention include molecules of the RAIDD induced cell death pathwaywhich interact with a RAIDD polypeptide.

[0087] Further antagonist according to the present invention includeboth the RAIDD death and protease domains and fragments of each. Suchforms of the RAIDD protein, which may be naturally occurring orsynthetic, antagonize RAIDD polypeptide mediated activity by competingfor binding to cellular ligands of RAIDD. Thus, such antagonists includefragments of the RAIDD polypeptide containing ligand binding domains.

[0088] RAIDD polypeptide antagonists also include small molecules whichbind to and occupy active regions of the RAIDD polypeptides therebymaking the polypeptide inaccessible to ligands which bind thereto suchthat normal biological activity is prevented. Examples of smallmolecules include but are not limited to nucleotide sequences and smallpeptides or peptide-like molecules. Such molecules may be produced andscreened for activity by a variety of methods (e.g., Light and Lerner,Bioorganic & Medicinal Chemistry 3(7):955-967 (1995); Cheng et al., Gene171:1-8 (1996); Gates et al., J. Mol. Biol. 255:373-386 (1996)).

[0089] Other assays for identifying potential antagonists and agonistsof RAIDD induced cell death are described in the art and in Example 4.These assays include in vitro and in vivo binding assays for theidentification of molecules which interact with the RAIDD polypeptidesof the present invention, as described in Example 4, and the yeasttwo-hybrid system (Fields and Song, Nature 340:245-246 (1989)). Thesemethod involves contacting the RAIDD polypeptide of the presentinvention with a candidate molecule and determining whether themolecules interact. Thus, in an additional aspect, the present inventionis directed to a method for identifying molecules capable of binding theRAIDD polypeptides.

[0090] In a preferred embodiment, a RAIDD polypeptide is produced with ahistidine tag allowing for affinity purification usingnickel-nitrilo-tri-acetic acid (“Ni-NTA”) affinity resin. The His-RAIDDpolypeptide is purified and incubated with a candidate molecule whichhas been radiolabelled. The RAIDD polypeptide is then re-purified undernon-denaturing conditions and a determination is made as to whether thecandidate molecule co-purifies with the RAIDD polypeptide. When thecandidate molecule is a protein, this determination can be made byboiling the putative RAIDD-candidate molecule conjugate in an aqueoussolution with SDS followed by SDS-PAGE and autoradiography. Candidatemolecules which interact with the RAIDD polypeptide are potentialagonists and antagonist of RAIDD activity.

[0091] Having generally described the invention, the same will be morereadily understood by reference to the following examples, which areprovided by way of illustration and are not intended as limiting.

Examples Example 1 Expression and Purification of RAIDD in E. coli

[0092] The bacterial expression vector pQE9 (pD10) is used for bacterialexpression in this example. (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth,Calif., 91311). pQE9 encodes ampicillin antibiotic resistance(“Amp^(r)”) and contains a bacterial origin of replication (“ori”), anIPTG inducible promoter, a ribosome binding site (“RBS”), six codonsencoding histidine residues that allow affinity purification usingnickel-nitrilo-tri-acetic acid (“Ni-NTA”) affinity resin sold by QIAGEN,Inc., supra, and suitable single restriction enzyme cleavage sites.These elements are arranged such that an inserted DNA fragment encodinga polypeptide expresses that polypeptide with the six His residues(i.e., a “6×His tag”) covalently linked to the amino terminus of thatpolypeptide.

[0093] The DNA sequence encoding the desired portion of the RAIDDprotein is amplified from the deposited cDNA clone using PCRoligonucleotide primers which anneal to the amino terminal sequences ofthe desired portion of the RAIDD protein and to sequences in thedeposited construct 3′ to the cDNA coding sequence. Additionalnucleotides containing restriction sites to facilitate cloning in thepQE9 vector are added to the 5′and 3′primer sequences, respectively.

[0094] For cloning the protein, suitable 5′ primers would be apparent tothose skilled in the art. In a preferred embodiment, a primer at least15 to 20 nucleotides in length is used containing one or moreappropriately located restriction sites followed by at least 10nucleotides complementary to the amino terminal sequence encoding theRAIDD protein shown in SEQ ID NO:1. One of ordinary skill in the artwould appreciate, of course, that the point in the protein codingsequence where the 5′ primer begins may be varied to amplify a DNAsegment encoding any desired portion or domain of the complete RAIDDprotein.

[0095] Suitable 3′ primers would also be apparent to those skilled inthe art and include those preferably at least 15 to 20 nucleotides inlength, containing one or more appropriately located restriction sites,followed by at least 10 nucleotides complementary to either the carboxyterminal coding region or the non-coding region of the sequence shown inSEQ ID NO:1.

[0096] The amplified RAIDD DNA fragment and the vector pQE9 are digestedwith appropriate restriction enzymes to generate suitable “sticky” endsand the digested DNA molecules are then ligated together. Insertion ofthe RAIDD DNA into the restricted pQE9 vector can be done to place theRAIDD protein coding region downstream from the IPTG-inducible promoterand in-frame with an initiating AUG and the six histidine codons.

[0097] The ligation mixture is transformed into competent E. coli cellsusing standard procedures such as those described in Sambrook et al.,Molecular Cloning: a Laboratory Manual, 2nd Ed.; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989). E. coli strainM15/rep4, containing multiple copies of the plasmid pREP4, whichexpresses the lac repressor and confers kanamycin resistance(“Kan^(r)”), is used in carrying out the illustrative example describedherein. This strain, which is only one of many that are suitable forexpressing RAIDD protein, is available commercially from QIAGEN, Inc.,supra. Transformants are identified by their ability to grow on LBplates in the presence of ampicillin and kanamycin. Plasmid DNA isisolated from resistant colonies and the identity of the cloned DNAconfirmed by restriction analysis, PCR and DNA sequencing.

[0098] Clones containing the desired constructs are grown overnight(“O/N”) in liquid culture in LB media supplemented with both ampicillin(100 μg/ml) and kanamycin (25 μg/ml). The O/N culture is used toinoculate a large culture, at a dilution of approximately 1:25 to 1:250.The cells are grown to an optical density at 600 nm (“OD600”) of between0.4 and 0.6. Isopropyl-b-D-thiogalactopyranoside (“IPTG”) is then addedto a final concentration of 1 mM to induce transcription from the lacrepressor sensitive promoter, by inactivating the lacI repressor. Cellssubsequently are incubated further for 3 to 4 hours. Cells then areharvested by centrifugation.

[0099] The cells are then stirred for 3-4 hours at 4° C. in 6 Mguanidine-HCl, pH 8. The cell debris is removed by centrifugation, andthe supernatant containing the RAIDD is loaded onto anickel-nitrilo-tri-acetic acid (“NiNTA”) affinity resin column(available from QIAGEN, Inc., supra). Proteins with a 6×His tag bind tothe NI-NTA resin with high affinity and can be purified in a simpleone-step procedure (for details see: The QIAexpressionist, 1995, QIAGEN,Inc., supra). Briefly the supernatant is loaded onto the column in 6 Mguanidine-HCl, pH 8, the column is first washed with 10 volumes of 6 Mguanidine-HCl, pH 8, then washed with 10 volumes of 6 M guanidine-HCl pH6, and finally the RAIDD is eluted with 6 M guanidine-HCl, pH 5.

[0100] The purified protein is then renatured by dialyzing it againstphosphate buffered saline (PBS) or 50 mM Na-acetate, pH 6 buffer plus200 mM NaCl. Alternatively, the protein can be successfully refoldedwhile immobilized on the Ni-NTA column. The recommended conditions areas follows: renature using a linear 6 M-1 M urea gradient in 500 mMNaCl, 20% glycerol, 20 mM Tris/HCl pH 7.4, containing proteaseinhibitors. The renaturation should be performed over a period of 1.5hours or more. After renaturation the proteins can be eluted by theaddition of 250 mM imidazole. Imidazole is removed by a final dialyzingstep against PBS or 50 mM sodium acetate pH 6 buffer plus 200 mM NaCl.The purified protein is stored at 4° C. or frozen at −80° C.

[0101] Alternatively, a procedure similar to that described above may beemployed except that the bacterial expression vector pQE60 (QIAGEN,Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311) is used. The use ofsuitable primers with this vector will result in the covalent linkage ofa 6×His tag to the carboxyl terminus of the expressed polypeptide.Similarly, suitable vectors and primers may be used which will result inthe expression of a RAIDD polypeptide without any His tag.

Example 2 Cloning and Expression of RAIDD Protein in a BaculovirusExpression System

[0102] In this illustrative example, the plasmid shuttle vector pA2 GPis used to insert the cloned DNA encoding the protein into a baculovirusto express the RAIDD protein, using a baculovirus leader and standardmethods as described in Summers et al., A Manual of Methods forBaculovirus Vectors and Insect Cell Culture Procedures, TexasAgricultural Experimental Station Bulletin No.1555 (1987). Thisexpression vector contains the strong polyhedrin promoter of theAutographa californica nuclear polyhedrosis virus (AcMNPV) followed bythe secretory signal peptide (leader) of the baculovirus gp67 proteinand convenient restriction sites such as BamHI, Xba I and Asp718. Thepolyadenylation site of the simian virus 40 (“SV40”) is used forefficient polyadenylation. For easy selection of recombinant virus, theplasmid contains the beta-galactosidase gene from E. coli under controlof a weak Drosophila promoter in the same orientation, followed by thepolyadenylation signal of the polyhedrin gene. The inserted genes areflanked on both sides by viral sequences for cell-mediated homologousrecombination with wild-type viral DNA to generate viable virus thatexpresses the cloned polynucleotide.

[0103] Many other baculovirus vectors could be used in place of thevector above, such as pAc373, pVL941 and pAcIM1, as one skilled in theart would readily appreciate, as long as the construct providesappropriately located signals for transcription, translation, secretionand the like, including a signal peptide and an in-frame AUG asrequired. Such vectors are described, for instance, in Luckow et al.,Virology 170:31-39.

[0104] The cDNA sequence encoding the RAIDD protein in the depositedclone, is amplified using PCR oligonucleotide primers corresponding tothe 5′ and 3′ sequences of the gene.

[0105] Suitable primers for cloning the RAIDD protein can be designed asdescribed above in Example 1.

[0106] The amplified fragment is isolated from a 1% agarose gel using acommercially available kit (“Geneclean,” BIO 101 Inc., La Jolla,Calif.). The fragment then is digested with the appropriate restrictionenzymes and again is purified on a 1% agarose gel. This fragment isdesignated herein “F1”.

[0107] The plasmid is also digested with the appropriate restrictionenzymes and optionally, can be dephosphorylated using calf intestinalphosphatase, using routine procedures known in the art. The DNA is thenisolated from a 1% agarose gel using a commercially available kit(“Geneclean” BIO 101 Inc., La Jolla, Calif.). This vector DNA isdesignated herein “V1”.

[0108] Fragment F1 and the dephosphorylated plasmid V1 are ligatedtogether with T4 DNA ligase. E. coli HB101 or other suitable E. colihosts such as XL-1 Blue (Stratagene Cloning Systems, La Jolla, Calif.)cells are transformed with the ligation mixture and spread on cultureplates. Bacteria are identified that contain the plasmid with the humanRAIDD gene using the PCR method, in which one of the primers that isused to amplify the gene and the second primer is from well within thevector so that only those bacterial colonies containing the RAIDD genefragment will show amplification of the DNA. The sequence of the clonedfragment is confirmed by DNA sequencing. This plasmid is designatedherein pBacRAIDD.

[0109] Five μg of the plasmid pBacRAIDD is co-transfected with 1.0 μg ofa commercially available linearized baculovirus DNA (“BaculoGold mbaculovirus DNA”, Pharmingen, San Diego, Calif.), using the lipofectionmethod described by Felgner et al., Proc. Natl. Acad. Sci. USA84:7413-7417 (1987). 1 μg of BaculoGold™ virus DNA and 5 μg of theplasmid pBacRAIDD are mixed in a sterile well of a microtiter platecontaining 50 μl of serum-free Grace's medium (Life Technologies Inc.,Gaithersburg, Md.). Afterwards, 10 μl Lipofectin plus 90 μl Grace'smedium are added, mixed and incubated for 15 minutes at roomtemperature. Then the transfection mixture is added drop-wise to Sf9insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with1 ml Grace's medium without serum. The plate is rocked back and forth tomix the newly added solution. The plate is then incubated for 5 hours at27° C. After 5 hours the transfection solution is removed from the plateand 1 ml of Grace's insect medium supplemented with 10% fetal calf serumis added. The plate is put back into an incubator and cultivation iscontinued at 27° C. for four days.

[0110] After four days the supernatant is collected and a plaque assayis performed, as described by Summers and Smith, supra. An agarose gelwith “Blue Gal” (Life Technologies Inc., Gaithersburg) is used to alloweasy identification and isolation of gal-expressing clones, whichproduce blue-stained plaques. (A detailed description of a “plaqueassay” of this type can also be found in the user's guide for insectcell culture and baculovirology distributed by Life Technologies Inc.,Gaithersburg, page 9-10). After appropriate incubation, blue stainedplaques are picked with the tip of a micropipettor (e.g., Eppendorf).The agar containing the recombinant viruses is then resuspended in amicrocentrifuge tube containing 200 μl of Grace's medium and thesuspension containing the recombinant baculovirus is used to infect Sf9cells seeded in 35 mm dishes. Four days later the supernatants of theseculture dishes are harvested and then they are stored at 4° C. Therecombinant virus is called V-RAIDD.

[0111] To verify the expression of the RAIDD gene, Sf9 cells are grownin Grace's medium supplemented with 10% heat inactivated FBS. The cellsare infected with the recombinant baculovirus V-RAIDD at a multiplicityof infection (“MOI”) of about 2. Six hours later the medium is removedand is replaced with SF900 II medium minus methionine and cysteine(available from Life Technologies Inc., Rockville, Md.). If radiolabeledproteins are desired, 42 hours later, 5 μCi of ³⁵S-methionine and 5 μCi³⁵S-cysteine (available from Amersham) are added. The cells are furtherincubated for 16 hours and then they are harvested by centrifugation.The proteins in the supernatant as well as the intracellular proteinsare analyzed by SDS-PAGE followed by autoradiography (if radiolabeled).Microsequencing of the amino acid sequence of the amino terminus ofpurified protein may be used to determine the amino terminal sequence ofthe mature protein and thus the cleavage point and length of thesecretory signal peptide.

Example 3 Cloning and Expression of RAIDD in Mammalian Cells

[0112] A typical mammalian expression vector contains the promoterelement, which mediates the initiation of transcription of mRNA, theprotein coding sequence, and signals required for the termination oftranscription and polyadenylation of the transcript. Additional elementsinclude enhancers, Kozak sequences and intervening sequences flanked bydonor and acceptor sites for RNA splicing. Highly efficienttranscription can be achieved with the early and late promoters fromSV40, the long terminal repeats (LTRS) from Retroviruses, e.g., RSV,HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV).However, cellular elements can also be used (e.g., the human actinpromoter). Suitable expression vectors for use in practicing the presentinvention include, for example, vectors such as PSVL and PMSG(Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC37146) and pBC12MI (ATCC 67109). Mammalian host cells that could be usedinclude, human HeLa 293, H9 and Jurkat cells, mouse NIH3T3 and C127cells, Cos 1, Cos 7 and CV 1, quail QC1-3 cells, mouse L cells andChinese hamster ovary (CHO) cells.

[0113] Alternatively, the gene can be expressed in stable cell linesthat contain the gene integrated into a chromosome. The co-transfectionwith a selectable marker such as dhfr, gpt, neomycin, or hygromycinallows the identification and isolation of the transfected cells.

[0114] The transfected gene can also be amplified to express largeamounts of the encoded protein. The DHFR (dihydrofolate reductase)marker is useful to develop cell lines that carry several hundred oreven several thousand copies of the gene of interest. Another usefulselection marker is the enzyme glutamine synthase (GS) (Murphy et al.,Biochem J. 227:277-279 (1991); Bebbington et al., Bio/Technology10:169-175 (1992)). Using these markers, the mammalian cells are grownin selective medium and the cells with the highest resistance areselected. These cell lines contain the amplified gene(s) integrated intoa chromosome. Chinese hamster ovary (CHO) and NSO cells are often usedfor the production of proteins.

[0115] The expression vectors pC1 and pC4 contain the strong promoter(LTR) of the Rous Sarcoma Virus (Cullen et al., Molecular and CellularBiology, 438447 (March, 1985)) plus a fragment of the CMV-enhancer(Boshart et al., Cell 41:521-530 (1985)). Multiple cloning sites, e.g.,with the restriction enzyme cleavage sites BamHI, XbaI and Asp718,facilitate the cloning of the gene of interest. The vectors contain inaddition the 3′ intron, the polyadenylation and termination signal ofthe rat preproinsulin gene.

Example 3(a) Cloning and Expression in COS Cells

[0116] The expression plasmid, pRAIDD HA, is made by cloning a cDNAencoding RAIDD into the expression vector pcDNAI/Amp or pcDNAIII (whichcan be obtained from Invitrogen, Inc.).

[0117] The expression vector pcDNAI/amp contains: (1) an E. coli originof replication effective for propagation in E. coli and otherprokaryotic cells; (2) an ampicillin resistance gene for selection ofplasmid-containing prokaryotic cells; (3) an SV40 origin of replicationfor propagation in eukaryotic cells; (4) a CMV promoter, a polylinker,an SV40 intron; (5) several codons encoding a hemagglutinin fragment(i.e., an “HA” tag to facilitate purification) followed by a terminationcodon and polyadenylation signal arranged so that a cDNA can beconveniently placed under expression control of the CMV promoter andoperably linked to the SV40 intron and the polyadenylation signal bymeans of restriction sites in the polylinker. The HA tag corresponds toan epitope derived from the influenza hemagglutinin protein described byWilson et al., Cell 37:767 (1984). The fusion of the HA tag to thetarget protein allows easy detection and recovery of the recombinantprotein with an antibody that recognizes the HA epitope. pcDNAIIIcontains, in addition, the selectable neomycin marker.

[0118] A DNA fragment encoding the RAIDD protein is cloned into thepolylinker region of the vector so that recombinant protein expressionis directed by the CMV promoter. The plasmid construction strategy is asfollows. The RAIDD cDNA of the deposited clone is amplified usingprimers that contain convenient restriction sites, much as described inExample 1 above for construction of vectors for expression of RAIDD inE. coli. Suitable primers would be apparent to one skilled in the art.In a preferred embodiment such primers are at least 15 to 20 nucleotidesin length and have the following characteristics. The 5′ primer containsone or more appropriately located restriction sites, a Kozak sequence,an AUG start codon and 5 to 10 codons of the 5′ coding region of theRAIDD sequence shown in SEQ ID NO:1. The 3′ primer contains one or moreappropriately located restriction sites, a stop codon, and 15 to 20 bpof either 3′ coding or noncoding sequence shown in SEQ ID NO:1. Theseprimers may be designed such that any region or domain of the RAIDDprotein is cloned.

[0119] The PCR amplified DNA fragment and the vector, pcDNAI/Amp, aredigested with appropriate restriction enzymes and then ligated. Theligation mixture is transformed into E. coli strain SURE (available fromStratagene Cloning Systems, 11099 North Torrey Pines Road, La Jolla,Calif. 92037), and the—transformed culture is plated on ampicillin mediaplates which then are incubated to allow growth of ampicillin resistantcolonies. Plasmid DNA is isolated from resistant colonies and examinedby restriction analysis or other means for the presence of theRAIDD-encoding fragment.

[0120] For expression of recombinant RAIDD, COS cells are transfectedwith an expression vector, as described above, using DEAE-DEXTRAN, asdescribed, for instance, in Sambrook et al., Molecular Cloning: aLaboratory Manual, Cold Spring Laboratory Press, Cold Spring Harbor,N.Y. (1989). Cells are incubated under conditions for expression ofRAIDD by the vector.

[0121] Expression of the RAIDD-HA fusion protein is detected byradiolabeling and immunoprecipitation, using methods described in, forexample Harlow et al., Antibodies: A Laboratory Manual, 2nd Ed.; ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1988). To thisend, two days after transfection, the cells are labeled by incubation inmedia containing ³⁵S-cysteine for 8 hours. The cells and the media arecollected, and the cells are washed and lysed with detergent-containingRIPA buffer: 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% DOC, 50 mM TRIS, pH7.5, as described by Wilson et al., cited above. Proteins areprecipitated from the cell lysate and from the culture media using anHA-specific monoclonal antibody. The precipitated proteins then areanalyzed by SDS-PAGE and autoradiography. An expression product of theexpected size is seen in the cell lysate, which is not seen in negativecontrols.

Example 3(b) Cloning and Expression in CHO Cells

[0122] The vector pC4 is used for the expression of RAIDD protein.Plasmid pC4 is a derivative of the plasmid pSV2-dhfr (ATCC Accession No.37146). The plasmid contains the mouse DHFR gene under control of theSV40 early promoter. Chinese hamster ovary- or other cells lackingdihydrofolate activity that are transfected with these plasmids can beselected by growing the cells in a selective medium (alpha minus MEM,Life Technologies) supplemented with the chemotherapeutic agentmethotrexate. The amplification of the DHFR genes in cells resistant tomethotrexate (MTX) has been well documented (see, e.g., Alt, F. W.,Kellems, R. M., Bertino, J. R., and Schimke, R. T., 1978, J Biol. Chem.253:1357-1370, Hamlin, J. L. and Ma, C. 1990, Biochem. et Biophys. Acta,1097:107-143, Page, M. J. and Sydenham, M. A. 1991, Biotechnology9:64-68). Cells grown in increasing concentrations of MTX developresistance to the drug by overproducing the target enzyme, DHFR, as aresult of amplification of the DHFR gene. If a second gene is linked tothe DHFR gene, it is usually co-amplified and over-expressed. It isknown in the art that this approach may be used to develop cell linescarrying more than 1,000 copies of the amplified gene(s). Subsequently,when the methotrexate is withdrawn, cell lines are obtained whichcontain the amplified gene integrated into one or more chromosome(s) ofthe host cell.

[0123] Plasmid pC4 contains for expressing the gene of interest thestrong promoter of the long terminal repeat (LTR) of the Rous SarcomaVirus (Cullen et al., Molecular and Cellular Biology, March1985:438-447) plus a fragment isolated from the enhancer of theimmediate early gene of human cytomegalovirus (CMV) (Boshart et al.,Cell 41:521-530(1985)). Downstream of the promoter are BamHI, XbaI, andAsp718 restriction enzyme cleavage sites that allow integration of thegenes. Behind these cloning sites the plasmid contains the 3′ intron andpolyadenylation site of the rat preproinsulin gene. Other highefficiency promoters can also be used for the expression, e.g., thehuman β-actin promoter, the SV40 early or late promoters or the longterminal repeats from other retroviruses, e.g., HIV and HTLVI.Clontech's Tet-Off and Tet-On gene expression systems and similarsystems can be used to express the RAIDD in a regulated way in mammaliancells (Gossen, M., & Bujard, H. 1992, Proc. Natl. Acad. Sci. USA 89:5547-5551). For the polyadenylation of the mRNA other signals, e.g.,from the human growth hormone or globin genes can be used as well.Stable cell lines carrying a gene of interest integrated into thechromosomes can also be selected upon co-transfection with a selectablemarker such as gpt, G418 or hygromycin. It is advantageous to use morethan one selectable marker in the beginning, e.g., G418 plusmethotrexate.

[0124] The plasmid pC4 is digested with the appropriate restrictionenzyme(s) and then dephosphorylated using calf intestinal phosphatase byprocedures known in the art. The vector is then isolated from a 1%agarose gel.

[0125] The DNA sequence encoding the complete RAIDD protein is amplifiedusing PCR oligonucleotide primers corresponding to the 5′ and 3′sequences of the gene. Suitable primers would be apparent to one skilledin the art. Such primers would be at least 15 to 20 nucleotides inlength. Preferably, suitable 5′ primers would contain one or moreappropriately located restriction sites followed by an efficient signalfor initiation of translation in eukaryotes, as described by Kozak, M.,J. Mol. Biol. 196:947-950 (1987), and 15 to 20 bases of the codingsequence of the RAIDD shown in SEQ ID NO:1. Similarly, suitable 3′primers would contain one or more appropriately located restrictionsites followed by 15 to 20 nucleotides complementary to thenon-translated region of the RAIDD gene shown in SEQ ID NO:1. Theseprimers may be designed such that any region or domain of the RAIDDprotein is cloned.

[0126] The amplified fragment is digested with the appropriaterestriction enzyme(s) and then purified again on a 1% agarose gel. Theisolated fragment and the dephosphorylated vector are then ligated withT4 DNA ligase. E. coli HB101 or XL-1 Blue cells are then transformed andbacteria are identified that contain the fragment inserted into plasmidpC4 using, for instance, restriction enzyme analysis.

[0127] Chinese hamster ovary cells lacking an active DHFR gene are usedfor transfection. 5 μg of the expression plasmid pC4 is cotransfectedwith 0.5 μg of the plasmid pSV2-neo using lipofectin (Felgner et al.,supra). The plasmid pSV2neo contains a dominant selectable marker, theneo gene from Tn5 encoding an enzyme that confers resistance to a groupof antibiotics including G418. The cells are seeded in alpha minus MEMsupplemented with 1 mg/ml G418. After 2 days, the cells are trypsinizedand seeded in hybridoma cloning plates (Greiner, Germany) in alpha minusMEM supplemented with 10, 25, or 50 ng/ml of metothrexate plus 1 mg/mlG418. After about 10-14 days single clones are trypsinized and thenseeded in 6-well petri dishes or 10 ml flasks using differentconcentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM).Clones growing at the highest concentrations of methotrexate are thentransferred to new 6-well plates containing even higher concentrationsof methotrexate (1 μM, 2 μM, 5 μM, 10 mM, 20 mM). The same procedure isrepeated until clones are obtained which grow at a concentration of100-200 μM. Expression of the desired gene product is analyzed, forinstance, by SDS-PAGE and Western blot or by reverse phase HPLCanalysis.

Example 4 RAIDD, A Novel Death Adaptor Molecule

[0128] A sequence (I.M.A.G.E. Consortium CloneID 109053) was identifiedin the NCBI GenBank™ EST database as having statistically significanthomology (p<0.001) to the prodomain of the human ICE-like proteaseICH-1. This EST clone was used to isolate a full length cDNA thatencoded a protein of 199 amino acids with a predicted molecular mass of22 kDa (SEQ ID NO:2). Given its sequence and interactions (see below),this molecule was designated RAIDD (for RIP-Associated ICH-1/CED-3homologous protein with a Death Domain) (SEQ ID NO:2). A BLAST searchrevealed that this was a novel molecule possessing two domains that haddistinct homologies. Residues 8-80 displayed statistically significanthomology (p<0.001) with the prodomains of the human ICE-like proteaseICH-1 (SEQ ID NO:3) and the C. elegans death protease CED-3 (SEQ IDNO:4) (FIG. 2B). Residues 123-194 encoded a C terminal Death Domain (DD)with significant homology to other DD-containing adaptor molecules (SEQID NOs:5, 6 and 7) (FIG. 2C). Northern blot analysis revealed RAIDD tobe constitutively expressed as a 1.35 kb transcript in all adult andfetal tissues examined with substantial expression in adult heart,testis, liver, skeletal muscle, fetal liver and kidney.

[0129] The homology between the N terminal domain (NTD) of RAIDD and theprodomains of ICH-1 and CED-3 suggested that RAIDD likely binds ICH-1and CED-3 through a homophilic mechanism using this homologousinteraction domain. This notion was investigated by examining theinteraction of ³⁵S-labeled mammalian ICE/ced-3 family members withimmobilized RAIDD in vitro. In keeping with the hypothesis, RAIDDspecifically associated with ICH-1 but not with the other mammalianICE-like proteases tested (i.e., ICE, TX, ICE-LAP6, ICE-LAP3, Yama andMch2α). To confirm whether this binding was mediated by a NTD-prodomaininteraction, deletion mutants of ICH-1 and RAIDD were utilized. ICH-1bound a truncated form of RAIDD encoding only the NTD (amino acidresidues 1-117 in SEQ ID NO:2), but failed to associate with a versionencoding just the DD (amino acid residues 95-199 in SEQ ID NO:2).Conversely, ICH-1 without the prodomain (amino acid residues 141-436 ofICH-1) failed to associate with RAIDD while the prodomain alone (aminoacid residues 1-140 of ICH-1) bound well. These results weresubsequently confirmed by in vitro testing where transfected 293 celllysates were immunoprecipitated for RAIDD (Flag epitope-tagged) andassociating molecules detected by immunoblotting. To ask if an analogousinteraction occurred with CED-3, 293 cells were cotransfected withexpression constructs encoding CED-3 and RAIDD or a truncated form ofRAIDD lacking the NTD. RAIDD, but not its deleted counterpart,associated with CED-3. Taken together, these results argue for a modelin which the NTD by a homophilic mechanism selectively associates withICE/ced-3 family members by binding to the prodomain.

[0130] To further characterize the NTD-prodomain interaction, bindingstudies were performed to test the association of wild type moleculeswith corresponding ³⁵S-labeled point mutants within the prodomain ofICH-1 and CED-3 or the NTD of RAIDD (FIG. 4). Mutations were introducedin residues highly conserved in all three molecules. More important,however, were alterations in residues Leu₂₇ and Gly₆₅ in the prodomainof CED-3 as these correspond to inactivating mutations of the ced-3 genein C. elegans (n1040 and n718) (Ellis, H. M. & Horvitz, H. R., Cell44:817-829 (1986); Shaham, S. & Horvitz, H. R., Genes and Development10:578-591 (1996)). Additionally, residues corresponding to Leu₂₇ andGly₆₅ in CED-3 were also mutated in RAIDD and ICH-1 (FIG. 4). In allinstances where Leu₂₇ and Gly₆₅ were mutated, binding was abolished,highlighting the importance of these residues in mediating theprodomain-NTD interaction (FIG. 4). It is therefore tempting tospeculate that just such a disruption might be the biochemical basis forthe CED-3 inactivating mutations (n1040 and n718). Mutation of conservedhydrophobic residues in the ICH-1 prodomain including F₈₂, F₈₅ and L₈₉(mt7, mt5 and mt3) abolished binding to the NTD of RAIDD. Surprisingly,it took the mutation of both conserved negatively charged residues (D₈₃and E₈₇, mt8) to eliminate binding (FIG. 4).

[0131] Having established interactions undertaken by the NTD of RAIDD,we asked which DD-containing molecules were engaged by the DD of RAIDD.Initially, we examined all five known mammalian DD-containing proteinsimplicated in apoptosis (Cleveland, J. L. & Thle, J. N., Cell 81:479-482(1995)). As RAIDD did not directly bind in vitro to either of the twoDD-containing receptors (Fas or TNFR-1) (data not shown), we asked if itmight bind to the DD-containing receptor associated molecules (FADD,TRADD, or RIP). RAIDD specifically bound RIP but not FADD or TRADD invivo. However, in the concomitant presence of RIP, RAIDD was able tocomplex with TRADD. Since RIP is a component of the TNFR-1 signalingcomplex (Hsu, H. et al., Immunity 4:387-396 (1996)), it was conceivablethat it could recruit RAIDD to the signaling complex. RAIDD, in turn,would be positioned to recruit ICH-1 and thereby establish a directphysical link to the effector ICE/CED-3-like death proteases. To testwhether such a relay of signaling proteins could indeed be assembled,293 cells were transiently transfected with expression vectors encodingTNFR-1, DD-containing adaptor molecules (TRADD, RIP, RAIDD, FADD),ICH-1, and ICE as a control. As anticipated, TNFR-1 complexed with RAIDDin the presence of TRADD and RIP, and through RAIDD, ICH-1 was recruitedto the signaling complex. Next, we asked whether RAIDD, besides engagingthe death pathway, could also induce NF-kB activation since its upstreampartner, RIP, is capable of engaging both pathways (Hsu, H. et al.,Immunity 4:387-396 (1996)). Transfected RAIDD did not activate an NF-kBreporter construct (data not shown), consistent with a primary role inapoptosis. These results suggest that RAIDD can potentially function asan adaptor molecule recruiting the death protease ICH-1 to the TNFR-1signaling complex.

[0132] To address the functional role of RAIDD in apoptosis, MCF-7 celllines were transiently transfected with a RAIDD expression construct.Transfected cells underwent an apoptotic demise which was inhibitable bythe broad spectrum ICE peptide inhibitor z-VAD-fmk, a poxvirus encodedserpin known to inhibit apoptosis, CrmA and catalytically inactive ICH-1that presumably behaved as a dominant negative by competing outendogenous ICH-1 (FIG. 5). The ability of dominant negative ICH-1 toblock RAIDD-induced cell death gave functional credence to thebiochemical interaction. However, neither mutant ICH-1 or variousputative dominant negative versions of RAIDD were able to block TNFR-1induced cell death (data not shown) presumably due to the unimpededfunctioning of the alternate TRADD-FADD-FLICE death pathway.

[0133] It is unclear why two seemingly redundant pathways would bedeployed by a single receptor, but differential cell or tissue specificutilization remain a distinct possibility. More importantly, howeverthese studies illuminate how prodomain interactions can impart bindingspecificity, a prerequisite for the assembly of a functioning deathmachine.

[0134] Methods

[0135] cDNA Cloning. Sequence corresponding in the pro-domain of ICH-1was used to screen the NCBI GenBank™ EST database using establishedexpressed-sequence-tag methods. Several overlapping clones wereidentified as having statistically significant homology (p<0.001) to thepro-domain of ICH-1. One such clone, I.M.A.G.E. Consortium CloneID109053 (Lennon, G. et al., Genomics 33:151-152 (1996)) was characterizedfurther and subjected to both automated and manual DNA sequencing.Additionally, a human K562 cell line cDNA library (Dr. John Lowe,University of Michigan) was screened with a ³²P-labeled Pac 1/EcoR1restriction fragment of the EST clone (Sambrook et al., supra).Hybridizing clones were characterized by automated DNA sequencing.Sequence assembly, comparison and alignment were performed using DNASTARsoftware.

[0136] Northern Blot analysis. Adult and fetal human multiple tissueNorthern blots (Clonetech, Palo Alto, Calif.) were hybridized accordingto the manufacturer's instructions with the same ³²P-labeled probe usedfor library screening.

[0137] Expression Vectors. Mammalian cell expression vectors encodingAU1-FADD, FLAG-TNFR1, Myc-TRADD and Myc-RIP have been describedpreviously (Hsu, H. et al., Immunity 4:387-396 (1996); Chinnaiyan, A. M.et al., Cell 81:505-12 (1995)). AU1-RAIDD or FLAG-RAIDD was generated byPCR using custom oligonucleotide primers encoding the AU1 or FLAGepitope. Amplified fragments were cloned into the mammalian expressionvector pcDNA3 (Invitrogen). In-frame deletion mutants were generated byPCR with or without an N-terminal AU1 epitope and subsequently subclonedinto pcDNA3. Point mutations were created by site-directed mutagenesisusing a two-step PCR method (Higuchi, R. et al., Nucleic Acids Research16:7251-7367 (1988)) and confirmed by sequence analysis. Catalyticallyinactive ICH-1 was created by mutating the active site cysteine₃₀₂ toalanine.

[0138] In Vitro Binding Assay. Constructs encoding His-tagged proteins(FADD-DN, RAIDD, RAIDD-N (amino acid residues 1-117), RAIDD-DD (aminoacid residues 95-199)) were prepared in the prokaryotic expressionvector pET23b (Novagen). The tagged proteins were expressed, purified,and immobilized into Ni²⁺ beads according to standard methodology. TheGST-Fas and TNFR-1 fusion proteins were prepared as described previously(Chinnaiyan, A. M. et al., Cell 81:505-12 (1995)). ³⁵S-labeled proteinswere obtained by in vitro transcription/translation using the TNTT7-coupled reticulocyte lysate system (Promega).

[0139] Following translation, equivalent amounts of ³⁵S-labeled proteins(ICE, TX, ICE-LAP6, ICE-LAP3, ICH-1, Δpro-ICH-1 (amino acid residues141-436), pro-ICH-1 (amino acid residues 1-140), Yama and Mch2α) wereincubated with His-tagged proteins or GST-fusion proteins immobilizedinto Ni²⁺ beads or glutathione beads, respectively, as described inprior publications (Chinnaiyan, A. M. et al., Cell 81:505-12 (1995);Muzio, M. et al., Cell 85:817-827 (1996)). Beads were washed, boiled inSDS sample buffer, eluted proteins resolved by SDS-PAGE and visualizedfollowing autoradiography.

[0140] Alternatively, ICH-1 protein was produced in human 293 embryonickidney cells transfected with the AU1-ICH-1 expression construct.

[0141] In Vivo Binding Assay. 293 cells were transfected withcombinations of expression constructs encoding FLAG-RAIDD and one of thefollowing proteins: AU1-ICH-1, AU1-pro-ICH-1 (amino acids 141-436) orAU1-ICE as a control. FLAG-CED-3 was cotransfected with either the deathdomain of RAIDD (AU1-tagged, amino acids 123-199) or the full lengthmolecule (AU1-RAIDD). Cleared cell lysates were immunoprecipitated usinganti-FLAG antibody and analyzed by immunoblotting with either anti-AU1MAb or anti-RAIDD antipeptide antibody (produced in response to asynthetic peptide comprising amino acid residues 99 to 117 in SEQ IDNO:2).

[0142] Transfection, Coimmunoprecipitation and Western Analysis. 293cells were transiently transfected with indicated plasmids, lysed in 1ml lysis buffer (50 mM Tris [pH 7.6], 150 mM NaCl, 0.1% NP-40) andincubated either with mouse control IgG or anti-FLAG MAb. Immunecomplexes were precipitated by the addition of protein G-Sepharose(Sigma). Following extensive washing, the Sepharose beads were boiled insample buffer eluted proteins were processed as described previously(O'Rourke, K. M. et al., J. of Biol. Chem. 267:24921-24924 (1992)).

[0143] On Overexpressed RAIDD and ICH-1 are Recruited to the TNFR1Signaling Complex. 293 cells were transfected with expression vectorsexpressing either Flag-TNFR1 or Flag-RAIDD and one or more of thefollowing proteins: myc-RIP, myc-TRADD, AU1-FADD, AU1-ICE and AU1-ICH1.Detergent extracts were immunoprecipitated with either control mouse IgGor anti-FLAG MAb. Co-precipitating proteins were analyzed byimmunoblotting with anti-epitope tag antibody (anti-myc and anti-AU1anti-AU1 or RAIDD antipeptide antibody).

[0144] Apoptosis Assay. MCF-7 human breast carcinoma cells, CrmAexpressing stable cell lines (Tewari, M. & Dixit, V. M., J. Biol. Chem.270:3255-60 (1995)), or 293-EBNA cells were transiently transfected with0.25 μg of the reporter plasmid pCMV β-galactosidase plus 1.5 μg of testplasmid encoding either FADD or RAIDD. The broad spectrum ICE familyinhibitor z-VAD-fmk (Enzyme Systems Products) was added to the cells ata concentration of 20 μm, 5 hours following transfection. Forty eighthours later, the assay was performed as described previously(Chinnaiyan, A. M. et al., Cell 81:505-12 (1995)).

[0145] It will be clear that the invention may be practiced otherwisethan as particularly described in the foregoing description andexamples.

[0146] Numerous modifications and variations of the present inventionare possible in light of the above teachings and, therefore, are withinthe scope of the appended claims.

[0147] The entire disclosure of all publications (including patents,patent applications, journal articles, laboratory manuals, books, orother documents) cited herein are hereby incorporated by reference.

1 7 1 1160 DNA Artificial Sequence Isolated Homo sapiens cDNA 1aacagtcagg attccggttg cagtttttct cccccgcccc aaagatacgt ggttgcagac 60ggagaa atg gag gcc aga gac aaa caa gta ctc cgc tca ctt cgc ctg 108 MetGlu Ala Arg Asp Lys Gln Val Leu Arg Ser Leu Arg Leu 1 5 10 gag ctg ggtgca gag gta ttg gtg gag gga ctg gtt ctt cag tac ctc 156 Glu Leu Gly AlaGlu Val Leu Val Glu Gly Leu Val Leu Gln Tyr Leu 15 20 25 30 tac cag gaagga atc ttg acg gaa aac cat att caa gaa atc aat gct 204 Tyr Gln Glu GlyIle Leu Thr Glu Asn His Ile Gln Glu Ile Asn Ala 35 40 45 caa acc aca ggcctc cgg aaa aca atg ctc ctg ctg gat atc cta cct 252 Gln Thr Thr Gly LeuArg Lys Thr Met Leu Leu Leu Asp Ile Leu Pro 50 55 60 tcc agg ggc cct aaagca ttt gat aca ttc cta gat tcc cta cag gag 300 Ser Arg Gly Pro Lys AlaPhe Asp Thr Phe Leu Asp Ser Leu Gln Glu 65 70 75 ttt ccc tgg gtc agg gagaag ctg aag aag gca agg gaa gag gcc atg 348 Phe Pro Trp Val Arg Glu LysLeu Lys Lys Ala Arg Glu Glu Ala Met 80 85 90 acc gac ctg cct gca ggt gacaga ttg act ggg atc ccc tcg cac atc 396 Thr Asp Leu Pro Ala Gly Asp ArgLeu Thr Gly Ile Pro Ser His Ile 95 100 105 110 ctc aac agc tcc cca tcagac cgg cag att aac cag ctg gcc cag agg 444 Leu Asn Ser Ser Pro Ser AspArg Gln Ile Asn Gln Leu Ala Gln Arg 115 120 125 ctg ggc cct gag tgg gagccc atg gtg ttg tct ctg gga ctg tcc cag 492 Leu Gly Pro Glu Trp Glu ProMet Val Leu Ser Leu Gly Leu Ser Gln 130 135 140 acg gat atc tac cgc tgtaag gcc aac cac ccc cac aac gtg cag tcg 540 Thr Asp Ile Tyr Arg Cys LysAla Asn His Pro His Asn Val Gln Ser 145 150 155 cag gtg gtg gag gcc ttcatc cgt tgg cgg cag cgc ttc ggg aag cag 588 Gln Val Val Glu Ala Phe IleArg Trp Arg Gln Arg Phe Gly Lys Gln 160 165 170 gcc acc ttc cag agc ctgcac aac ggg ctg cgg gct gtg gag gtg gac 636 Ala Thr Phe Gln Ser Leu HisAsn Gly Leu Arg Ala Val Glu Val Asp 175 180 185 190 ccc tcg ctg ctc ctgcac atg ttg gag tgatggtgcc tccagcaacc 683 Pro Ser Leu Leu Leu His MetLeu Glu 195 gctggggagt gtgtccctga gtcatgtggg ctgaatcctg actttcactcagagcaggtg 743 gttttttgtg taggtttgtt ttttattttt gatgatcttc agatggaaggagaaaacagg 803 gtttccacta gacattactt gaaaggccag attactcagc agatctcccatgttggctca 863 acaattcttt gtttttaatt gcttgaagat tgcattgttg taattgttcagtttttaaat 923 gtgtaatggc attttaatag actagtaaat cacagtggtt ccaaatatatacccatatat 983 atatatatcc atatatatat ctcatgtcat cacattacag gcaggtgtctcatatgtaaa 1043 acatttacct gaatgttgtc tgaggactga actgtggact ttactattcataatgataaa 1103 ataataaaat gcgaattact ataaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaa 1160 2 199 PRT Homo sapiens 2 Met Glu Ala Arg Asp Lys Gln ValLeu Arg Ser Leu Arg Leu Glu Leu 1 5 10 15 Gly Ala Glu Val Leu Val GluGly Leu Val Leu Gln Tyr Leu Tyr Gln 20 25 30 Glu Gly Ile Leu Thr Glu AsnHis Ile Gln Glu Ile Asn Ala Gln Thr 35 40 45 Thr Gly Leu Arg Lys Thr MetLeu Leu Leu Asp Ile Leu Pro Ser Arg 50 55 60 Gly Pro Lys Ala Phe Asp ThrPhe Leu Asp Ser Leu Gln Glu Phe Pro 65 70 75 80 Trp Val Arg Glu Lys LeuLys Lys Ala Arg Glu Glu Ala Met Thr Asp 85 90 95 Leu Pro Ala Gly Asp ArgLeu Thr Gly Ile Pro Ser His Ile Leu Asn 100 105 110 Ser Ser Pro Ser AspArg Gln Ile Asn Gln Leu Ala Gln Arg Leu Gly 115 120 125 Pro Glu Trp GluPro Met Val Leu Ser Leu Gly Leu Ser Gln Thr Asp 130 135 140 Ile Tyr ArgCys Lys Ala Asn His Pro His Asn Val Gln Ser Gln Val 145 150 155 160 ValGlu Ala Phe Ile Arg Trp Arg Gln Arg Phe Gly Lys Gln Ala Thr 165 170 175Phe Gln Ser Leu His Asn Gly Leu Arg Ala Val Glu Val Asp Pro Ser 180 185190 Leu Leu Leu His Met Leu Glu 195 3 69 PRT Homo sapiens 3 Leu Lys LysAsn Arg Val Val Leu Ala Lys Gln Leu Leu Leu Ser Glu 1 5 10 15 Leu LeuGlu His Leu Leu Glu Lys Asp Ile Ile Thr Leu Glu Met Arg 20 25 30 Glu LeuIle Gln Ala Lys Val Gly Ser Phe Ser Gln Asn Val Glu Leu 35 40 45 Leu AsnLeu Leu Pro Lys Arg Gly Pro Gln Ala Phe Asp Ala Phe Cys 50 55 60 Glu AlaLeu Arg Glu 65 4 68 PRT Caenorhabditis elegans 4 Leu Glu Arg Asn Ile MetMet Phe Ser Ser His Leu Lys Val Asp Glu 1 5 10 15 Ile Leu Glu Val LeuIle Ala Lys Gln Val Leu Asn Ser Asp Asn Gly 20 25 30 Asp Met Ile Asn SerCys Gly Thr Val Arg Glu Lys Arg Arg Glu Ile 35 40 45 Val Lys Ala Val GlnArg Arg Gly Asp Val Ala Phe Asp Ala Phe Tyr 50 55 60 Asp Ala Leu Arg 655 74 PRT Homo sapiens 5 Ile Cys Asp Asn Val Gly Lys Asp Trp Arg Arg LeuAla Arg Gln Leu 1 5 10 15 Lys Val Ser Asp Thr Lys Ile Asp Ser Ile GluAsp Arg Tyr Pro Arg 20 25 30 Asn Leu Thr Glu Arg Val Arg Glu Ser Leu ArgIle Trp Lys Asn Thr 35 40 45 Glu Lys Glu Asn Ala Thr Val Ala His Leu ValGly Ala Leu Arg Ser 50 55 60 Cys Gln Met Asn Leu Val Ala Asp Leu Val 6570 6 81 PRT Homo sapiens 6 Phe Ala Arg Ser Val Gly Leu Lys Trp Arg LysVal Gly Arg Ser Leu 1 5 10 15 Gln Arg Gly Cys Arg Ala Leu Arg Asp ProAla Leu Asp Ser Leu Ala 20 25 30 Tyr Glu Tyr Glu Arg Glu Gly Leu Tyr GluGln Ala Phe Gln Leu Leu 35 40 45 Arg Arg Phe Val Gln Ala Glu Gly Arg ArgAla Thr Leu Gln Arg Leu 50 55 60 Val Glu Ala Leu Glu Glu Asn Glu Leu ThrSer Leu Ala Glu Asp Leu 65 70 75 80 Leu 7 77 PRT Homo sapiens 7 Ile ArgGlu Asn Leu Gly Lys His Trp Lys Asn Cys Ala Arg Lys Leu 1 5 10 15 GlyPhe Thr Gln Ser Gln Ile Asp Glu Ile Asp His Asp Tyr Glu Arg 20 25 30 AspGly Leu Lys Glu Lys Val Tyr Gln Met Leu Gln Lys Trp Val Met 35 40 45 ArgGlu Gly Ile Lys Gly Ala Thr Val Gly Lys Leu Ala Gln Ala Leu 50 55 60 HisGln Cys Ser Arg Ile Asp Leu Leu Ser Ser Leu Ile 65 70 75

What is claimed is:
 1. An isolated nucleic acid molecule comprising apolynucleotide having a nucleotide sequence at least 95% identical to asequence selected from the group consisting of: (a) a nucleotidesequence encoding a polypeptide comprising amino acids from about 1 toabout 199 in SEQ ID NO:2; (b) a nucleotide sequence encoding apolypeptide comprising amino acids from about 2 to about 199 in SEQ IDNO:2; (c) a nucleotide sequence encoding a polypeptide comprising aminoacids from about 8 to about 80 in SEQ ID NO:2; (d) a nucleotide sequenceencoding a polypeptide comprising amino acids from about 123 to about194 in SEQ ID NO:2; (e) a nucleotide sequence encoding a polypeptidecomprising amino acids from about 99 to about 199 in SEQ I) NO:2; (f) anucleotide sequence encoding a polypeptide comprising amino acids fromabout 118 to about 199 in SEQ ID NO:2; (g) a nucleotide sequenceencoding a polypeptide having the amino acid sequence encoded by thecDNA clone contained in ATCC Deposit No. 97824; and (h) a nucleotidesequence complementary to any of the nucleotide sequences in (a), (b),(c), (d), (e), (f), or (g).
 2. An isolated nucleic acid moleculecomprising a polynucleotide which hybridizes under stringenthybridization conditions to a polynucleotide having a nucleotidesequence identical to a nucleotide sequence in (a), (b), (c), (d), (e),(f), (g), or (h) of claim 1 wherein said polynucleotide which hybridizesdoes not hybridize under stringent hybridization conditions to apolynucleotide having a nucleotide sequence consisting of only Aresidues or of only T residues.
 3. A method for making a recombinantvector comprising inserting an isolated nucleic acid molecule of claim 1into a vector.
 4. A recombinant vector produced by the method of claim3.
 5. A method of making a recombinant host cell comprising introducingthe recombinant vector of claim 4 into a host cell.
 6. A recombinanthost cell produced by the method of claim
 5. 7. A recombinant method forproducing a RAIDD polypeptide, comprising culturing the recombinant hostcell of claim 6 under conditions such that said polypeptide is expressedand recovering said polypeptide.
 8. An isolated RAIDD polypeptide havingan amino acid sequence at least 95% identical to a sequence selectedfrom the group consisting of: (a) amino acids from about 1 to about 199in SEQ ID NO:2; (b) amino acids from about 2 to about 199 in SEQ IDNO:2; (c) amino acids from about 8 to about 80 in SEQ ID NO:2; (d) aminoacids from about 123 to about 194 in SEQ ID NO:2; (e) amino acids fromabout 99 to about 199 in SEQ ID NO:2; (f) amino acids from about 118 toabout 199 in SEQ ID NO:2; (g) the amino acid sequence of the RAIDDpolypeptide having the amino acid sequence encoded by the cDNA clonecontained in ATCC Deposit No. 97824; and (h) the amino acid sequence ofan epitope-bearing portion of any one of the polypeptides of (a), (b),(c), (d), (e), (f), or (g).
 9. An isolated antibody that bindsspecifically to a RAIDD polypeptide of claim
 8. 10. A method foridentifying agonists and antagonists of RAIDD induced cell deathcomprising: (a) contacting cells which express a RAIDD polypeptide witha candidate molecule, and (b) measuring the level of RAIDD induced celldeath; whereby, an increased level of RAIDD induced cell death comparedto a standard level of RAIDD induced cell death indicates that saidcandidate molecule is an agonist and a decreased level of RAIDD inducedcell death compared to said standard indicates that said candidatemolecule is an antagonist.